This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Two nitrogenase MoFe protein mutants, resulting from the deletion of either the nifH or nifB gene, are being studied. Mutants of these types are iron-molybdenum cofactor (FeMoco) deficient, yet contain sufficient iron for an [8Fe-7S] P-cluster. Crystallographic data support the presence of a "normal" P-cluster, similar to that observed in wild-type MoFe protein, in the nifB- MoFe protein. EPR studies suggest that the cluster in the nifH- MoFe protein could be a P-cluster precursor. nifB- mutants can be directly activated in vitro by the addition of isolated FeMoco whereas nifH- mutants cannot. The electronic and structural difference between these two mutants have been studied using Fe K-edge x-ray absorption edge and extended fine structure (EXAFS) analyses, which confirmed the presence of a wild-type P-cluster in the nifB- MoFe protein and a novel [Fe4S4]-based cluster in the nifH- MoFe protein. In a related set of experiments, it was shown that incubation with NifH does not change the structure of the cluster in the nifH- mutant, although it does enable modest nitrogenase activity after FeMoco insertion. This pre-incubated nifH- MoFe protein is the first example of a functioning MoFe protein without an intact P-cluster.